Behavioral observations show that clinically significant depressive symptoms are rare accompaniments of induced hypogonadism in these healthy premenopausal women. Additionally, neither night-time hot flushes nor disturbed sleep are sufficient to cause depressive symptoms in hypogonadal young women (reported in MH002537-23 DIRP: Psychobiology And Treatment Of Perimenopausal Mood Disorders). Thus this paradigm serves as an excellent comparison group for the women with reproductive endocrine-related mood disorders who undergo identical hormone manipulations. While hypothalamic-pituitary-adrenal (HPA) axis dysregulation frequently accompanies depression, the role of ovarian steroids in the regulation of HPA axis responsivity in humans is not well-characterized. To evaluate HPA axis regulation in women, we performed combined dexamethasone-suppression/CRH-stimulation tests and 24-hour urinary free cortisol (UFC) levels in 25 healthy asymptomatic women during each of the three hormone conditions created in this hormone manipulation protocol. We observed that progesterone significantly increased the secretion of HPA axis hormones compared with both estradiol and hypogonadism. Thus, progesterone but not estradiol up-regulates HPA axis function in women. In our earlier studies of regional cerebral blood flow using PET, we reported for the first time in humans that induced hypogonadism was associated with the elimination of the normal pattern of cortical activation in the dorsolateral prefrontal cortex (DLPFC); whereas both estradiol and progesterone replacement restored the normal pattern of cortical activation during a working memory task. We are pursuing our original findings that ovarian steroids modulate prefrontal cortical activity in women by augmenting older gold-standard imaging techniques (i.e., O15 PET), in which the technology is relatively stable over time and the activation task paradigms are kept relatively constant over the long-term course of these studies, with newer hypothesis-driven, cutting edge task paradigms and analytic approaches (i.e., fMRI). We employ several different activational paradigms as well as resting state measures during the neuroimaging procedures that will allow us to test neural circuits and interregional functional connectivity relevant to the phenomenology of affective disorders (i.e., prefrontal cortical function, reward system). We have completed studies in over 50 asymptomatic women. Results from our O15 PET studies, demonstrate that estradiol significantly increases resting regional cerebral blood flow (rCBF) activity in regions of the default networks: medial prefrontal cortex (mPFC) and posterior cingulate compared with the hypogonadal state; whereas, progesterone significantly increases resting rCBF in the medial orbital frontal cortex (mOFC) and putamen compared with the hypogonadal state. During N-back activation, activation of the DLPFC is increased during both estradiol and progesterone compared with hypogonadism, and ovarian steroids (or their absence) modulates functional connectivity between the DLPFC and hippocampus. Specifically, hormone condition had significant effects on the direction of hippocampal functional connectivity with both left and right DLPFC: the expected working memory-related negative correlation was observed during estradiol add-back and during progesterone add-back there was a positive hippocampal-right DLPFC correlation but there was no significant correlation during ovarian suppression with Lupron alone (i.e., hypogonadism. These data demonstrate that ovarian steroids modulate hippocampal-PFC functional connectivity, consistent with a role for these hormones in modulating the hippocampus at the cellular level. Interestingly, our findings indicate that progesterone and estradiol have opposite effects on this in vivo measure of hippocampal-DLPFC cooperativity, substantiating the complex influence of gonadal steroids in brain circuitry and calling for further clinical and preclinical investigation. Moreover, preliminary data using a navigational (spatial memory) task we observed greater hippocampal activity in the presence of estradiol compared with hypogonadism, consistent with substantial pre-clinical evidence that estrogen enhances hippocampal synaptic plasticity. Preclinical effects of progesterone on hippocampal function have been less clear, but our preliminary data also indicate that progesterone, like estradiol, affects hippocampal activity during spatial navigation in humans. Our findings that estradiol and progesterone regulate activities within mPFC, mOFC, and hippocampus have implications for understanding the potential impacts of both estradiol and progesterone on affective-adaptation and stress-responsivity. Preclinical studies have demonstrated that these brain areas are sites at which the effects of sex and sex-steroids modulate the effects of stress on brain function and behavior. Finally, recent data has documented the influence of genotype on cognitive performance and affective-adaptation. We now have identified for the first time in humans significant interactions between sex steroids and variations in specific genes regulated by ovarian steroids (i.e., catechol-o-methyl transferase (COMT) and brain-derived neurotrophic factor (BDNF)) on measures of task-activated regional cerebral blood flow. Estradiol mitigates the effect of COMT genotype on DLPFC activation during the N-back task. While interactions between steroid hormones and BDNF are well documented on the molecular level, there is little behavioral and neural circuitry data in humans. We now document that women carrying the less physiologically active BDNF met allele experience increased hippocampal activation during the N-back task compared with carriers of the val allele. These data in humans compliment similar data in the genetically altered met knock-in mouse that show a differential behavioral (more anxious) phenotype during estradiol exposure. These data suggest both brain regional and genomic substrates in which changes in gonadal steroids could modulate affective state, and are consistent with preclinical studies that document the modulatory effects of ovarian steroids on behaviors and brain regions, as well as a growing literature demonstrating the importance of both genotypic variation and these same brain regions in depression. Finally, we have measured grey matter (GM) volume using structural MRI scans across the menstrual cycle and under pharmacologically controlled hormonal conditions. GM volume in the hippocampus increased during the luteal compared with the follicular phase of the menstrual cycle and during progesterone addback compared with both estradiol and hypogonadism There has been a widespread presumption in clinical neuroscience that ovarian steroids regulate 5HT transport function leading to sex and sex steroid-related differences in affective adaptation. We employ the 5HT transporter ligand DASB during both naturalistic and experimentally-induced conditions to examine claims that ovarian steroids are important physiological regulators of the 5HT transporter. We have completed a study of 5HT transport function across the menstrual cycle, and are about to complete a similar study examining 5HT transporter function during pharmacologically controlled hormonal conditions. These data will be the first to characterize the effects of sex steroids on5HT transporter function in the human brain, and will either confirm or seriously question the physiologic relevance of the widely embraced assumption that ovarian steroid-related modulations of 5HT function underlie both many of the observed sex-differences in affective disorders and reproductive endocrine-related mood disorders including postpartum and perimenopausal depressions.